Study of the molecular mechanism involved in recycling of matrix protein receptor, PEX5, during glycosome biogenesis in Trypanosoma brucei
(2012)
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- Authors
- Supervisors
- Michels, Paul
- Abstract
- (en) Trypanosoma brucei is the parasitic protist that is responsible for human sleeping sickness in Africa, a disease for which no adequate, affordable and harmless treatment is available. The parasite lives as a so-called procyclic-form trypanosome in the midgut of the tsetse fly, the vector that transmits the trypanosomes between people. It has been previously established that glycolysis is essential for the bloodstream form of the parasite, its life-cycle stage in humans, hence representing a promising drug target. Glycolysis in trypanosomatids is compartmentalized in peroxisome-like organelles called glycosomes, a unique feature not found in cells of other eukaryotes. Biogenesis of peroxisomes, organelles found in most eukaryotes, and of glycosomes in trypanosomatids are mediated by homologous proteins called peroxins (acronym PEX). The peroxins of trypanosomes are considered potentially good drug targets not only because glycolysis is essential for the parasites, but also because trypanosomes die when proper compartmentalization of glycolytic enzymes within glycosomes is disrupted. The import of proteins into the glycosomal matrix involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS-1) present at the C-terminus of the majority of these proteins. In yeasts and mammalian cells it has previously been shown that the cargo-loaded PEX5 associates with the peroxisomal membrane, delivers its cargo and is then ubiquitinated, a modification that serves as a signal for retrieval of PEX5 from the organelle to be used for further cycles of import (monoubiquitination) or, when recycling is impaired, for its proteasome-dependent degradation (polyubiquitination). We have found stable monoubiquitinated PEX5 in cytosolic fractions of wild-type bloodstream- and procyclic-form T. brucei. This modification appeared to be resistant to DTT, suggesting the conjugation of an ubiquitin moiety to a lysine residue of PEX5. We reason that this modified PEX5 species represents recycled molecules that have been efficiently exported by the recycling peroxin complex and that are in transit to be deubiquitinated, as a physiological step in the receptor cycle. We have identified the T. brucei orthologue of PEX4, the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast. This protein is expressed in both bloodstream and procyclic forms and is associated with the cytosolic face of the glycosomal membrane, probably via its interaction with the putative TbPEX22 that we also identified. Creation of a ∆PEX4 procyclic cell line by deletion of both alleles of the TbPEX4 gene enabled us to demonstrate that this peroxin is involved in TbPEX5 monoubiquitination. Surprisingly, after transfection of this mutant with a construct for expression of the Green Fluorescent Protein with a PTS-1 followed by subcellular localization studies by live cell imaging and fluorescence microscopy, only a minor defect in glycosomal matrix protein import was observed. Analysis of the ∆PEX4 mutant by qPCR showed that other enzymes of the putative UBC repertoire were upregulated. We assume that another trypanosome-specific UBC protein has taken over the function of PEX4 in the monoubiquitination of PEX5, but with less efficiency. Interestingly, important defects in morphology and motility were found in the majority of the ∆PEX4 cells. We hypothesize that these seemingly glycosome-biogenesis unrelated defects resulting from the knockout of the TbPEX4 gene could be explained by a disregulation of the expression of UBCs that are orthologous to yeast and human UBCs involved in cell-cycle control. We have also observed that PEX5 ubiquitination was affected in procyclic cell lines in which TbPEX12 and TbPEX6 expression was decreased by RNA interference. These data thus indicate the involvement of these proteins in TbPEX5 cycling, comparable to what has previously been demonstrated for their counterparts in yeasts and mammalian cells, but nonetheless with some differences as suggested by our studies. TbPEX12 and TbPEX6 were shown to be necessary for correct glycosomal matrix protein import and for cell viability, proving that the TbPEX5 recycling process is also essential. In addition, we have investigated the presence of channel-forming activities in glycosomal membrane preparations of T. brucei bloodstream forms. We found that glycosomal membrane proteins, when reconstituted in planar lipid bilayers, gave rise to the formation of three main kinds of channels. These were revealed by electrophysiological techniques. Currents were measured with amplitudes of 70-80, 20-25 and 8-11 pA (at a holding potential of +10 mV and with 3.0 M KCl as an electrolyte). The channels were in a fully open state over the membrane potential range +150 to -150 mV and showed no sub-conductance transitions. The channel with current amplitude of 20-25 pA is anion-selective while the other two types are slightly selective for cations. The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel’s pore. We discuss the relevance of these findings for the transport of glycolytic intermediates and other metabolites between the glycosomal lumen and cytosol. We propose different explanations for the apparent paradox of on one hand the presence of non-selective channels in the glycosomal membrane and on the other hand the experimentally proved low exchange of metabolites between the glycosomal matrix and cytosol. Importantly, the discovery of these non-selective channels in the glycosomal membrane of T. brucei may set new criteria for the size and physicochemical properties of inhibitors targeted to glycolytic and other enzymes inside glycosomes that will be designed as potential drugs for sleeping sickness.
- Affiliations
Citations
Gualdron Lopez, M. (2012). Study of the molecular mechanism involved in recycling of matrix protein receptor, PEX5, during glycosome biogenesis in Trypanosoma brucei. https://hdl.handle.net/2078.5/158374